Immunological agglutination reactions are used for identifying various kinds of blood types and for detecting various kinds of antibodies and antigens in blood samples and other aqueous solutions. In a conventional procedure, a sample of red blood cells is mixed with serum or plasma in test tubes or microplates, and the mixture may then be incubated and centrifuged. Various reactions either occur or do not occur depending on, for example, the blood type of the red blood cells or whether certain antibodies are present in the blood sample. Typically, these reactions manifest themselves as binding of cells or particles with antigens or antibodies on their surfaces, referred to as agglutinates. Thus, the absence of any such clumps indicates that no reaction has occurred; and the presence of such clumps indicates that a reaction has occurred, with the size and amount of such clumps being a quantitative indicator of the level or concentration in the sample, or an indicator of the reaction strength, affinity of the complex for which the blood sample was tested.
A traditional agglutination test method, referred to as column agglutination technology, is presently available (as disclosed in U.S. Pat. No. 5,594,808 which is hereby incorporated by reference in its entirety). Column agglutination technology is defined as the analysis of blood and blood products utilizing filtration as a means of separating agglutinated, precipitated, absorbed or adsorbed particulate components from non-reactive components for immunoassay applications. In this method, gel or glass bead microparticles are contained within a small column, referred to as a microcolumn. A reagent, such as anti-A, is dispensed in a diluent in the microcolumn and test red blood cells are placed in a reaction chamber above the column. The column, which is typically one of a multitude of columns formed in a transparent cassette, is centrifuged. The centrifuging accelerates the reaction, if any, by drawing the red blood cells through the reagent to enhance the agglutination, and subsequently urges any unbound cells to the bottom of the column. The glass beads or gel in the microcolumn act as a filter and resist or impede downward movement of the particles in the column. As a result, the nature and distribution of the particles in the microcolumn after centrifuging provides a visual indication of whether any agglutination reaction occurred in the microcolumn, and if so, of the strength of that reaction.
In particular, if no agglutination reaction occurs, then all or virtually all of the red blood cells in the microcolumn pass downward, during centrifuging, to the bottom of the column and form a pellet at that bottom. If there is a very strong reaction between the reagent and the red blood cells, virtually all of the red blood cells agglutinate, and large agglutinates form at the top of the microcolumn, above the gel or glass beads contained therein. The gel or glass beads prevent the agglutinates from passing, during centrifuging, to the bottom of the column, so that after centrifuging the agglutinates remain on the surface of the gel or beads.
If there is a reaction between the reagent and the blood cells, but this reaction is not as strong as the above-described very strong reaction, then some but not all of the red blood cells agglutinate. The percentage of red blood cells that agglutinate and the size of the agglutinated particles both vary directly with the strength of the reaction. During centrifuging, the unreacted blood cells pass to the bottom of the column, and the distance that the agglutinated particles pass downward through the column depends on the size and number of those particles. Hence, the size of the pellet of red blood cells at the bottom of the microcolumn, and the extent to which the agglutinates penetrate into the gel or glass beads in the microcolumn, are both inversely related to the strength of the reaction between the reagent and the red blood cells.
With some column agglutination technology analyzers, after the desired processing steps have been performed, the microcolumn is observed, or read, by a human operator, who then classifies the reaction between the reagent and the red blood cells. Conventionally, the reaction is classified as either negative or positive; and if positive, the reaction is then further classified into one of four classes depending on the strength of the reaction.
Conventional blood analysis systems (as disclosed in U.S. Pat. No. 5,620,898 which is hereby incorporated in its entirety by reference) include a multitude of stations or assemblies or subsystems, each of which performs one or more functions, and typically a significant amount of operator supervision and labor is needed to operate the systems. In this application, an imaging analysis subsystem is disclosed which allows a high quality image of a diagnostic cassette or an object of interest to be obtained which can be analyzed in an automated manner so that an appropriate analytical result is obtained without operator intervention. One automated method comprises the steps of producing an illuminated monochromatic digital image of the microcolumn on an array of pixels, and assigning to each pixel in the illuminated image, a data value representing the intensity of the illuminated image on the pixel. These data values are then processed according to a predetermined program to determine if an agglutination pattern is present and, if so, to classify that pattern into one of a plurality of predefined classes. Another automated method disclosed herein comprises the steps of producing multiple illuminated monochromatic images of the microcolumn on an array of pixels, and assigning to each pixel in the illuminated image, a data value representing the intensity of the illuminated image on the pixel. These data values are then processed according to a predetermined program to construct a color digital image which is then used to determine if an agglutination pattern is present and, if so, to classify that pattern into one of a plurality of predefined classes. In both of the processing procedures above, the pixel array is separated into a plurality of zones, and the data values for the pixels in each zone are processed according to a respective predetermined procedure to determine values for a predefined set of variables. Then, those determined values are processed to determine whether an agglutination pattern is present in the solution, and if so, to classify that pattern into one of the predefined classes.
The solutions are contained in a column having glass microbeads or gel. The image processing program searches the location of the column in the source image on the pixel array; and after the column is located, the program generates a window to cover the column where the red cells are located. The program then selects three reference regions from inside and outside the column and measures the intensity or gray levels in these regions, and these measured gray levels are used to determine certain threshold values that are subsequently used in the processing program.
For the feature calculation, the bead/gel column is divided into five different zones. The region on top of the bead/gel column is defined as the positive zone, the region at the bottom of the column is defined as the negative zone, and the area between the positive and negative zones is separated into three intermediate zones (also considered to be “positive zones”). The red cells located in the positive zones are extracted using a threshold method, and the red cell agglutinates located in the intermediate zones are extracted using a morphological filter. In addition, the balance of the red cells between the left and right sides of the column is determined. For each column, the above parameters are preferably calculated for both front and back side images of the column, and the two calculated values for each parameter are combined. The agglutination reaction is then classified on the basis of these combined features. An algorithm for the evaluation of the column agglutination results is disclosed by Jian Shen, Mykola Yaremko, Rosemary Chachowski, Josef Atzler, Thierry Dupinet, Daniel Kittrich, Hansjoerg Kunz, Karl Puchegger, and Reiner Rohlfs in U.S. Pat. No. 5,594,808 entitled “Method and system for classifying agglutination reactions” and by Jian Shen, Mykola Yaremko, Rosemary Chachowski, Josef Atzler, Thierry Dupinet, Daniel Kittrich, Hansjoerg Kunz, Karl Puchegger, and Reiner Rohlfs in U.S. Pat. No. 5,768,407 entitled “Method and system for classifying agglutination reactions” both of which are hereby incorporated by reference in their entirety.
Some of the advantages of the invention disclosed herein lies in the use of a gray-scale digital camera or sensor as compared to a conventional color digital camera or sensor which require the application of superimposed color filter arrays. Color filter arrays (as disclosed in U.S. Pat. No. 3,971,065 which is hereby incorporated in its entirety by reference), when producing a color digital image of a specified output spatial resolution, require the use of interpolation algorithms which reduces the effective resolution of the color digital image that results. The reduced resolution color digital image so produced causes a decrease in the specificity of the column agglutination algorithm described above making the outcome of the test less certain. Alternatively, one could employ a color digital camera or sensor that uses color filter arrays such that an output full resolution color digital image is produced. However, that would require a digital sensor having a physically larger sensor (more rows and columns of pixels) such that when used in conjunction with a color filter array and the associated interpolation algorithm that the color digital image so produced would be of equivalent full resolution. The drawback in using this latter method being increased cost and a longer optical imaging path, the longer imaging path being required to obtain an equivalent depth of field when compared to the invention disclosed herein.